Sperm-oolemma penetration (SOP) is fundamental to fertilization. However, in mammals, the molecular basis of this dynamic process is incompletely understood. The P.I. has mapped two tightly- linked mutations on mouse Chromosome (Chr) 17 in the t complex, Stop1p and Stop1d (Sperm-t complex-oolemma penetration 1 proximal and Sperm-t complex-oolemma penetration 1 distal, respectively) whose activity significantly decreases the ability of affected sperm from penetrating zona pellucida-free eggs (the "stop" phenotype). Because these genes offer a key to understanding the mechanisms that regulate the SOP process, the PI will determine if the "stop" phenotype derives from a defect in the binding and/or fusion phase(s) of SOP by testing sperm from mice expressing the "stop" phenotype in novel in vitro sperm-oolemma binding assays (Specific Aim I). Since Stop1d appears to be a major regulator of "stop" expression, and because Stop1d has been mapped only to a moderate resolution of approximately 3 centiMorgans (cM), the PI will map the Stop1d locus to high resolution (approximately 0.1-0.6 cM) by marker analysis of novel recombinant Chr 17 homologs from mice either expressing or not expressing the "stop" defect, and whose polymorphic breakpoints reside in the 3 cM Stop1d region (Specific Aim IIA). High resolution mapping will allow the PI to physically isolate both Stop1p and Stop1d loci in overlapping contiguous bacterial artificial chromosomes (BAC contigs) spanning each locus from proximal to distal flanking markers (Specific Aim IIB). In order to isolate candidate Stop1p and Stop1d genes, The PI will construct a testis-expression transcript map of each locus by differential display-PCR of mutant versus control testis mRNAs, and by cDNA selection of Stop1p and Stop1d BACs (Specific Aim IIIA). To determine which candidates are most likely to regulate "stop" activity, the PI will perform sequence and expression analysis of testis transcripts mapped to the Stop1p and Stop1d loci, testing for nucleic acid and presumed amino acid differences between allelic transcripts, and the tissue and temporal expression status of allelic transcripts (Specific Aim IIIB). The success of these experiments will contribute significantly to our understanding of a process, SOP, essential to fertilization, and will provide us with a model system for its further dissection.